CIB22 Antibody

Here’s a structured FAQ document addressing key research considerations for CIB22 antibody applications, synthesized from peer-reviewed methodologies and experimental data:

Advanced Research Questions

Can CIB22 antibody differentiate between Complex I assembly intermediates?

• Approach: Combine BN-PAGE (Blue Native PAGE) with immunoblotting using CIB22 antibody .

• Key findings:

  • CIB22 co-migrates with 980 kDa Complex I supercomplex in Arabidopsis .

  • Absence in cin22 mutants confirms subunit specificity .

How to address contradictory localization data in non-mitochondrial fractions?

• Hypothesis: Transient cytosolic expression during stress responses.

• Validation:

  • Fractionate cells under oxidative stress (e.g., 10 mM H₂O₂, 2 hr).

  • Compare mitochondrial/cytosolic distributions via subcellular fractionation .

Strategies for cross-species reactivity analysis beyond Arabidopsis

Methodological Best Practices

Quantitative analysis of CIB22 expression under abiotic stress

• Normalize signals to mitochondrial loading control (e.g., VDAC1).

• Use TOM20 antibody as a mitochondrial mass reference .

Antibody batch variability mitigation

• Pre-adsorb antibody with recombinant CIB22 protein (1 µg/mL, 1 hr incubation) .

• Validate each batch using standardized lysates from AT4G34700-overexpressing lines .

Data Contradiction Analysis

Discrepancies in molecular weight observations

• Scenario: Multiple bands detected at ~14 kDa and 28 kDa.

• Resolution:

  • 28 kDa band likely represents dimeric CIB22 (test via β-mercaptoethanol treatment) .

  • Confirm with reducing vs. non-reducing SDS-PAGE .

Conflicting subcellular localization reports

• Root cause: Antibody cross-reactivity with calcium-binding proteins (e.g., CIB2 in mammals) .

• Solution: CRISPR-Cas9-generated CIB22 null mutants as negative controls .